JP2003511047A - Methods for increasing transgenic biomass - Google Patents

Abstract

  (57) [Summary] The present invention provides (a) a step of breeding callus of a renewable plant comprising at least one genetic transformation phenomenon of interest; and (b) a callus comprising at least one genetic transformation phenomenon of interest. Arbitrarily selecting a step; (c) regenerating a whole transgenic plant called a primary transformant or a T0 plant from the callus of the plant; (d) pollinating the primary transformant with non-transgenic pollen. (E) recovering the resulting seed, designated T1, into which at least one transgene of interest has been inserted; (f) sowing said transgenic T1 and transforming the plants resulting therefrom into self-pollinated or free-pollinated plants. Pollinating each other; and (g) recovering T2 seeds. To provide a method for increasing the scan transgenic plant biomass.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention will be directed to methods for increasing the quantity and quality of transgenic biomass, and will be described and illustrated with particular reference to corn.

[0002]

[Prior art]

One factor that delays the formation of recombinant protein expression for therapeutic use in plants is the time required to obtain plant raw material containing the recombinant protein, which cannot be shortened.

This problem arises, in particular, when the time required to obtain the primary transformant is relatively long, on the other hand the organ in which the recombinant protein originates, in this case seed, is produced only in small quantities, especially in maize. Are most important in certain species such as. Thus, the use of this type of plant requires a breeding season prior to the purification and extraction of the recombinant protein produced. This breeding season is crucial in estimating the conversion process.

[0004]

[Problems to be Solved by the Invention]

The aim of the present invention is to increase the biomass obtained in the first generation and thus reduce the number of breeding seasons needed to obtain large amounts of biomass. This very significantly shortens the delay of research and development programs, and makes it possible to obtain the large quantities of plant raw material or biomass needed for the analysis of the purity of recombinant proteins of interest relatively inexpensively. .

Seed reproduction is a logarithmic phenomenon: in each generation there is about 100-fold amplification of the number of seeds obtained due to the classical transformed gene system, and its agricultural management value. Is low. Depending on the crossing method used, backcrossing or self-pollination, different proportions of the seeds formed will contain the gene of interest.

The Applicant has found that the answer to this problem lies in part in increasing the initial pool of seeds prior to sexual reproduction, thereby increasing the reproductive cycle and increasing the number of plants of the type described above. It has been found that it is possible to obtain a satisfactory amount of biomass and recombinant protein production for experiments in.

[0007]

[Means for Solving the Problems]

Thus, one object of the present invention is to include the following steps: (a) comprising at least one genetic transformation phenomenon of interest,
Reproducing a callus of a renewable plant; (b) optionally selecting a callus containing at least one genetic transformation phenomenon of interest; (c) a primary transformant or T0 plant from the callus of the plant Regenerating the entire transgenic plant referred to as; (d) pollinating the primary transformant with non-transgenic pollen; (e) inserting at least one transgene of interest, which may be referred to as T1 Collecting the seeds thus obtained; (f) sowing the transgenic T1 and pollinating the plants produced by self-pollination or free-pollination, respectively; and (g) collecting T2 seeds. A method for increasing transgenic plant biomass, which is characterized.

[0008] Preferably, the method described above also comprises the further step of performing a post-harvest phenotypic selection of the T2 seeds.

More preferably, the selection is T2 derived from plants used only as females.
Conducted on seeds.

[0010]

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the invention, transgenic T2 seeds have a different colored phenotype than non-transgenic seeds.

Particularly preferably, T2 seeds derived from plants used as male and female plants are recovered independently of each other.

[0012] Preferably said plant is allogenic and even more preferably said plant is maize.

In a preferred embodiment, the primary transformant is pruned or stamen excised before pollination with non-transgenic pollen.

In another preferred embodiment, the breeding process comprises several dozen, preferably about 20,
It consists of producing copy plants containing each genetic transformation phenomenon. For example, in the case of maize, a method of transforming maize using Agrobacterium by engaging immature embryos can be used to obtain maize callus containing one or more transgenes. This method involves a refolding period that produces transformants that can efficiently form copies. This copy formation is relatively easy to carry out, but makes it possible to obtain in vitro young transformed T0 plants which are strictly identical with respect to the transgene. These plants are visually isolated and form copies of early plants with excellent reliability. The additional work involved corresponds to cloning work, culturing all clones in phytotrons and then in the greenhouse, as well as controlling by molecular analysis, for example for the identification of valuable clones. This cloning will be performed on all primary transformants and then, for example, the most valuable ones (strongest expression according to insert purity) will be maintained after biochemical screening.

According to yet another preferred embodiment, transgenic T1 seeds are sown,
Cultured and used as a male plant.

Preferably the transgenic T1 seeds are sown in alternating rows, with non-transgenic plants as female plants, preferably 4/2 or 6/2.

More preferably in this case, the female non-transgenic plant is a sterile male plant.

According to a preferred variant of the above embodiment, the female non-transgenic plant is stamen excised.

Finally, preferably the female plants have a high agronomic value compared to the male plants.

The following detailed description illustrates preferred embodiments of the invention by way of non-limiting example.

[0021]

【Example】

Genetic transformation of plants requires the insertion of transgenes, selection of transformed cells, their proliferation, as well as their differentiation within neoplastic plants.

The method of genetically transforming maize, whether using a particle gun, is described in Agrobacteria as described by Y. Ishida et al. (Nature Biotech volume 14, June 1996, 745-749). Whether Um is used, this involves the growth of transformed callus with regenerative capacity. The first part of the invention is to amplify the number of regenerants obtained from the genetic transformation phenomenon.

Since cell biologists are able to grow in selective media, they are able to detect, sample and isolate each set of cells derived from the transformation phenomenon (untransformed tissues are In some cases can not grow).
Genetically modified neoplastic plants will then be formed through successive changes in the medium containing the appropriate photohormone balance. Biologists identify transgenic callus based on observation of the precise site formed. This macroscopic screening cannot separate the two genetic transformation phenomena occurring in adjacent cells. For this reason, biologists usually mature only one or two plants per identified callus, so that they do not carry the risk of various transformation phenomena originating separately from the same primary callus.

By carrying out a molecular analysis of DNA, it is possible to confirm whether the plants do in fact derive from the same transformation phenomenon. By performing Southerns with one or more probes directed against the transgene, judicious selection of restriction enzymes can be made to ensure that the transgene is actually inserted at the same location. is there. Experienced cell biologists have made good selections of callus and it has been empirically shown that regenerants originating from the same callus often derive from the same transformation phenomenon. Therefore, it is possible to increase the number of regenerants derived from the transformation phenomenon by adding one or two additional subculture steps during the conventional period of growth of transgenic callus. Further cell biology work is minimal, only about 3 in addition to methods that traditionally last on average 29 days.
Only the week prolongs the protocol. The Southern control as described above makes it possible to confirm that all the plants obtained actually derive from the same transformation phenomenon. For transgenes, they are copies but cell body clonal variations may occur.
It makes sense to consider producing about 20 copies of each transformation phenomenon in this way.

Commonly used methods for genetically transforming plants cannot completely control transgene insertion: insertion site selection, transgene copy number. Therefore, it is common to produce several dozen primary transformants using molecular constructs with the desired insertions in the plant genome, which allows the selection of optimal transformants. (For example, to confirm the presence of correct expression of the desired sequence only, or alternatively). Traditionally, these newly formed plants, called primary transformants,
After short-term growth in vitro, they are adapted and matured in the greenhouse. In the case of maize, the primary transformants are the most Generally pruned (stamens are removed). The progeny of the transgenic plant is
Obtained by pollinating transgenic ears with non-transgenic pollen. On the one hand, due to the variations of the maize used to carry out the genetic transformation, and on the other hand, the stress they undergo in vitro, the total number of T0 seeds obtained is almost 50 to 15
It is between 0. For the most common transgene single locus insertion,
Only 50% of the seeds formed with primary transformants (designated T1) are transgenic.

These seeds can then be used like any seed. If one of the transgenes confer resistance to the herbicide, it is easy to use this herbicide to select plants derived from transgenic seeds. Conventionally, these plants are either self-pollinated or freely pollinated to obtain T2 seeds. For this type of species suitable for in vitro culture, the degree of amplification by generation is approximately 100-fold. About 20 of each transformation phenomenon
The fact that copies are prepared means that about 1000 transgenic Ts per phenomenon
It is possible to obtain one seed (average 50 for the conventional method). This makes it possible to obtain a sufficient amount of pollen to consider pollinating non-transgenic plants with a minimum amount of work. This forms the second part of the invention. Cultures carried out in the open field or in the greenhouse are carried out in the same way as conventional production of maize hybrids. In this case, transgenic plants are used as males and are sown row by row (4/2 or most commonly 6/2 systems) alternating with non-transgenic plants used as females. These transgenic plants are ideally sterile male plants, which reduce the work, otherwise they are stamen excised. The plants are sown row by row rather than as a mixture, as it is necessary to be able to treat male plants with herbicides to remove those that do not inherit the transgene (50%). Furthermore, sowing in rows makes it possible to handle different early rice of male and female plants.

There are two advantages to this approach: -The amount of biomass is very significantly increased relative to the absence of female plant cultures. Two experiments in the field allowed us to reproduce 6 times as much biomass: more than 5 times more biomass was recovered when female plants were removed than when male plants were removed . -Biomass quality is much better because the plants used as females are hybrids with a higher additional agronomic value compared to male plants. Maize hybrids targeted for use with these transgenes can be used as female plants. Furthermore, from the second generation, a biomass is thus obtained which has a quality very close to that of future industrial biomass than would occur if the work were carried out only on male plants.

The proportion of transgenic seeds harvested from male plants is 75% in two ways (transgenic plant alone or hybrid type culture), while only 50% of seeds harvested from female plants are , Would be efficiently transgenic. To find a remedy to this, we propose to combine a gene conferring a phenotypic property that allows post-harvest industrial selection with the transgene of interest. This forms the third part of the invention. This can be done, for example, by modifying the color of corn seeds by modifying the enzymes involved in pigment biosynthesis. We have found that ourselves can carry out industrial sorting efficiently at very little cost by mixing different colored corns. After conditioning, it is possible to get a batch 95% or more pure by passing it through an industrial sorter once or twice. The screening can be carried out in the production of a few tons of seed, without any difficulty and at a very low cost, considering the machine used. Thus, the method is as follows: After hybrid-type culture, male and female plants are independently harvested and then selected for phenotypic properties. The product is thus completely transgenic.

The results are shown in Table 1, where the method of the present invention can be used to obtain 65 times more biomass with 1.33 times greater quality than the conventional method. It is shown. The term "quality" corresponds here to the proportion of transgenic seeds found in the recovered biomass. This represents an increase of about 3 weeks versus a total of 52 weeks (49 weeks without the present invention), resulting in a short-term additional delay at a slight additional cost.

If no addition of a gene conferring a phenotype specific to the gene of interest is desired, then it is sufficient to use only the first two parts of the invention. In this case, the obtained biomass is 120 times larger than the conventional method; for 1/6 biomass, the quality is similar to the conventional method except that the amount is 20 times greater,
For biomass, the quality is reduced by 1/3.

In Table 1, the values obtained are the average values taken as relative values for comparing the two methods. Amount: Number of corn seeds. Quality: A comparative proportion of transgenic seeds. With respect to genetic lines or backgrounds, hybrids produce seeds that are much closer to an industrial system than male transgenic plants.

[0032]

[Table 1]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (14)

[Claims] 1. A callus comprising (a) at least one genetic transformation phenomenon of interest and propagating a callus of a renewable plant; (b) a callus comprising at least one genetic transformation phenomenon of interest. (C) Regenerating an entire transgenic plant, called primary transformant or T0 plant, from the callus of the plant; (d) pollinating the primary transformant with non-transgenic pollen Step (e) collecting the resulting seed, designated T1, into which at least one transgene of interest has been inserted; (f) sowing the transgenic T1 and self-pollinating or free-pollinating the plant resulting therefrom. And (g) a step of collecting T2 seeds; , A method for increasing transgenic plant biomass. 2. A method according to claim 1, characterized in that it also comprises the further step of carrying out a post-harvest phenotypic selection of the T2 seeds. 3. A T2 derived from a plant whose selection is used only as a female.
Method according to claim 2, characterized in that it is carried out on seeds. 4. The method according to any one of claims 1 to 3, characterized in that the transgenic T2 seeds have a different colored phenotype than the non-transgenic seeds. 5. The method according to claim 1, characterized in that T2 seeds derived from plants used as male and female plants are recovered independently of each other. . 6. The plant according to claims 1 to 5, characterized in that the plant is allogenic.
The method according to any one of 1. 7. The plant according to claim 1, wherein the plant is corn.
7. The method according to claim 6. . 8. The primary transformant is characterized in that branches are cut or stamens are excised before pollination with non-transgenic pollen.
The method according to any one of claims 1 to 7. 9. The method according to claim 1, wherein the breeding step comprises producing a few dozen, preferably about 20, copy plants containing each genetic transformation phenomenon. The method according to one paragraph. 10. The method according to any one of claims 1 to 9, characterized in that the transgenic T1 seed is sown, cultivated and used as a male plant. 11. The transgenic T1 seeds are sown row by row alternating with non-transgenic plants as female plants, preferably 4/2 or 6/2. The method described. 12. The method according to claim 11, wherein the female non-transgenic plant is a sterile male plant. 13. Method according to claim 11, characterized in that the female non-transgenic plant is stamen excised. 14. The method according to any one of claims 11 to 13, characterized in that the female plant has a high farmland management value compared to the male plant.